Anti-pcsk9 compounds and methods for the treatment and/or prevention of cardiovascular diseases

ABSTRACT

Disclosed are compounds that modulate the physiological action of the proprotein convertase subtilisin kexin type 9 (PCSK9), and methods of using these modulators to reduce LDL-cholesterol levels and/or for the treatment and/or prevention of cardiovascular disease (CVD), including treatment of hypercholesterolemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/789,867, filed Mar. 15, 2013, the entirety of which is incorporatedherein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made with support from the National Heart,Lung and Blood Institute (NHLBI) under SBIR Grant No. HL092712. The U.S.Government has certain rights in this invention.

FIELD OF INVENTION

The present invention relates to compounds that modulate thephysiological action of the proprotein convertase subtilisin kexin type9 (PCSK9), including its interaction with the low density lipoproteinreceptor (LDLR). More specifically, the invention relates tocompositions comprising small molecule modulators of PCSK9 function andmethods of using these modulators as a medicament. The small moleculemodulators of PCSK9 function can be used therapeutically to lowerLDL-cholesterol levels in blood, and can be used in the preventionand/or treatment of cholesterol and lipoprotein metabolism disorders,including familial hypercholesterolemia, atherogenic dyslipidemia,atherosclerosis, and, more generally, cardiovascular disease (CVD).

BACKGROUND OF INVENTION

Cardiovascular diseases are the leading cause of death, withatherosclerosis being the leading cause of cardiovascular diseases.Atherosclerosis is a disease of the arteries and is responsible forcoronary heart disease associated with many deaths in industrializedcountries. Several risk factors for coronary heart disease have now beenidentified: dyslipidemia, hypertension, diabetes, smoking, poor diet,inactivity and stress. Dyslipidemia is elevation of plasma cholesterol(hypercholesterolemia) and/or triglycerides (TGs) or a low high-densitylipoprotein (HDL) level that contributes to the development ofatherosclerosis. It is a metabolic disorder that is proven to contributeto cardiovascular disease. In the blood, cholesterol is transported inlipoprotein particles, where the low-density lipoprotein (LDL)cholesterol (LDL-C) is considered “bad” cholesterol, whileHDL-cholesterol (HDL-C) is known as “good” cholesterol. Lipid andlipoprotein abnormalities are extremely common in the general populationand are regarded as a highly modifiable risk factor for cardiovasculardisease, due to the influence of cholesterol on atherosclerosis. Thereis a long-felt significant unmet need with respect to CVD with 60-70% ofcardiovascular events, heart attacks and strokes occurring despite thetreatment with statins (the current standard of care inatherosclerosis). Moreover, new guidelines suggest that even lower LDLlevels should be achieved in order to protect high risk patients frompremature CVD (1).

The establishment of a link between PCSK9 and cholesterol metabolism wasrapidly followed by the discovery that selected mutations in the PCSK9gene caused autosomal dominant hypercholesterolemia (2), suggesting thatthe mutations confer a gain-of-function (3) by increasing the normalactivity of PCSK9. This was supported by the experiment in whichwild-type and mutant PCSK9 (S127R and F216L) were expressed at highlevels in the livers of mice; hepatic LDLR protein levels felldramatically in mice receiving either the wild-type or mutant PCSK9 (4,5). No associated reductions in LDLR mRNA levels were observed,indicating that overexpression of PCSK9, whether mutant or wild-type,reduces LDLRs through a post-transcriptional mechanism.

Given that gain-of-function mutations in PCSK9 causehypercholesterolemia, it was reasonable to ask if loss-of-functionmutations would have the opposite effect and result inhypocholesterolemia. Three loss-of-function mutations in PCSK9 (Y142X,L253F, and C679X) were identified in African-Americans (6). Thesemutations reduce LDL-C levels by 28% and were shown to decrease thefrequency of CHD (defined as myocardial infarction, coronary death orcoronary revascularization) by 88%. Rashid et al. (7) studied themechanism of loss-of-function mutations in mice where PCSK9 wasinactivated. They reported that these knockout mice showed increasedhepatic LDLR protein (but not mRNA), increased clearance of circulatinglipoproteins and reduced plasma cholesterol levels. Structure-functionrelationship analysis of the naturally occurring mutations in PCSK9 hasalso provided insights into the mechanism of action of PCSK9.Interestingly, mutations in PCSK9 that were found to be associated withthe greatest reductions in LDL-C plasma levels are those that preventthe secretion of mature PCSK9 by disrupting its synthesis (Y142X),autocatalytic processing (L253F), or folding (C679X) (8). The Y142Xmutation produces no detectable protein because it occurs early in thetranscript and is predicted to initiate nonsense-mediated mRNA decay.Mutations in the catalytic domain (L253F) interfere with theautocatalytic cleavage of the protein. In cells expressing thePCSK9-253F, the amount of mature protein was reduced compared to that incells expressing PCSK9-WT, suggesting that the mutation inhibitsautocatalytic cleavage. The L253F mutation is near the catalytic triad(PCSK9 is a serine protease), therefore it might disrupt the active site(8). Inasmuch as autocatalytic cleavage of PCSK9 is required for exportof the protein out of the ER, the L253F mutation delays transport ofPCSK9 from the ER to the cell surface. The nonsense mutation (C679X) inPCSK9, which truncates the protein by 14 amino acids, did not interferewith protein processing, but the mature protein accumulates in the cellsand none is secreted, suggesting that the protein is cleaved normallybut is misfolded and is retained in the ER (8, 9).

The mechanism by which PCSK9 causes the degradation of the LDLR has notbeen fully elucidated. However, it is clear that the protease activityof PCSK9 is not required for LDLR degradation (10, 11). Li et al. (10)have co-expressed the prodomain and the catalytic domain in trans, andshowed that the secreted PCSK9 was catalytically inactive, yet it isfunctionally equivalent to the wild-type protein in lowering cellularLDL uptake and LDLR levels. Similar studies were also reported by McNuttet al. (11). Furthermore, Zhang et al. (12) has mapped PCSK9 binding tothe EGF-A repeat of the LDLR, and showed that such binding decreases thereceptor recycling and increases its degradation. They also reportedthat binding to EGF-A domain was calcium-dependent and increaseddramatically with reduction in pH from 7 to 5.2. Recently, Kwon et al.(13) determined the crystal structure of PCSK9 in complex with theLDLR-EGF-AB (EGF-A and EGF-B). The structure shows a well defined EGF-Adomain, but the EGF-B domain is disordered and absent from theirelectron density map. The EGF-A domain binds to the PCSK9 catalyticdomain at a site distant from the catalytic site, and makes no contactwith either the C-terminal domain or the prodomain (14).

Several strategies have been proposed for targeting PCSK9 (15). Strategy1: mRNA knockdown approaches include the use of antisenseoligonucleotides or RNAi. Antisense oligonucleotides administered tomice reduced PCSK9 expression by >90% and lowered plasma cholesterollevels by 53% (16). A single intravenous injection of an RNAi deliveredin lipidoid nanoparticles to cynomologous monkeys reduced plasma PCSK9levels by 70% and plasma LDL-C levels by 56% (17). Strategy 2: is toprevent binding of PCSK9 to the LDLR on the cell surface with a smallmolecule, a peptide, or an antibody directed against PCSK9. Adding EGF-Afragments to cultured cells inhibits the ability of exogenously addedPCSK9 to mediate LDLR degradation. Strategy 3: is to developsmall-molecule inhibitors of the PCSK9 processing. Despite evidence thatthe catalytic activity of PCSK9 is not required for LDLR degradation(11), an intracellular inhibitor of PCSK9 catalytic activity should beeffective, since autocatalytic processing of PCSK9 is required forsecretion of the protein from the ER. Following its synthesis, PCSK9undergoes an autocatalytic cleavage reaction that clips off theprodomain, but the prodomain remains attached to the catalytic domain(18, 19). The autocatalytic processing step is required for thesecretion of PCSK9 (20), likely because the prodomain serves as achaperone and facilitates folding. The continued attachment of theprodomain partially blocks the substrate binding pocket of PCSK9 (18,19). McNutt et al. (21) demonstrated that antagonism of secreted PCSK9increases LDLR expression in HepG2 cells. They show that anFH-associated LDLR allele (H306Y) that results in a gain-of-functionmutation is due to an increase in the affinity of PCSK9 to the LDLR,which would lead to enhanced LDLR destruction, and decreased plasmaLDL-C clearance. Furthermore, they were able to show elegantly thatblocking the secreted PCSK9 with LDLR (H306Y) subfragment resulted in anincrease in the level of LDLR in cultured HepG2 cells. Therefore, PCSK9acts as a secreted factor to cause LDLR degradation, and a smallmolecule inhibitor that interferes with the autocatalytic process shoulddecrease the amount of mature secreted PCSK9. This invention relates toidentification of small molecules that down-regulate the function ofPCSK9 using Strategy 3.

Recently (22-24), Regeneron/Sanofi and Amgen have reported Phase IIproof-of-concept data that validate the blocking of PCSK9 with amonoclonal antibody as a strategy for lowering LDL-C in patients notcontrolled on standard statin therapy. They reported that a singleinjection of their drug, called REGN727, slashed LDL levels by more than60% in clinical trial. Their approach follows Strategy 2 usingantibodies instead of small molecules. This Strategy 2 is also beingpursued by Merck, Novartis and Pfizer, while Strategy 1 is being pursuedby Alnylam, Idera and Santaris (25).

SUMMARY OF THE INVENTION

This invention relates to small molecules that selectively interact withand down modulate PCSK9 function. In a first embodiment, the agents usedin the practice of this invention have the general formula:

wherein R¹, R² and R³ are independently selected from the groupconsisting of H and optionally substituted lower alkyl, alkenyl,cycloalkyl, aryl, heterocycle, and heteroaryl;

X is O and NR⁴; R⁴ is selected from the group consisting of H and loweralkyl;

A is CO, CONR⁵, SO₂, C(═O)—O or a valence bond to R¹; R⁵ is selectedfrom the group consisting of H and lower alkyl;

M is CO and CR⁶R⁷; R⁶ and R⁷ are independently selected from the groupconsisting of H, lower alkyl;

Q is selected from the group consisting of O, and NR⁸; R⁸ is selectedfrom the group consisting of H, lower alkyl, or optionally when X is NR⁴and Q is NR⁸, R⁴ and R⁸ and the nitrogen atom to which each of R⁴ and R⁸is attached complete an optionally substituted 5- or 6-memberedheterocycle ring, as represented by —(CR_(a))_(n)—, wherein R_(a)represents H or lower alkyl and n=1 or 2; and the pharmaceuticallyacceptable salts and all stereoisomers of the compound. The compounds offormula I are believed to be novel compounds, with the exception of[(3-chloro-4-methylphenyl)carbamoyl](phenyl)methyl4-(carbamoylamino)benzoate.

In one embodiment, the present invention provides a method for thetreatment or prophylaxis of hypercholesterolemia, and/or at least onesymptom of dyslipidemia, atherosclerosis, CVD or coronary heart diseasein a patient in need of such treatment comprising administering to suchpatient a therapeutically effective amount of a compound of formula I,above.

In another embodiment, the present method for the treatment orprophylaxis of hypercholesterolemia, and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease in apatient in need of such treatment, is practiced by administering to apatient in need of such treatment or prophylaxis at least one of thefollowing compounds:

wherein X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent from the group consisting of hydroxyl,halogen, amino, alkoxy, carboxy, amido (including formamido, alkylamidoand arylamido), aminocarbonylamino, monoalkylaminocarbonylamino,dialkylaminocarbonylamino, carbamato, carboxamido,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkyl sulfonyl alkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl,dialkylaminosulfinyl and, optionally substituted, lower alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heterocycle, and heteroaryl;

X is O and NR⁴; R⁴ is selected from the group consisting of H and loweralkyl;

A is CO, CONR⁵, and SO₂; R⁵ is selected from the group consisting of Hand lower alkyl.

In alternative embodiments of the above-described method, a patient inneed of such treatment or prophylaxis is administered at least one ofthe following compounds:

wherein, in each of Formulas III, IV and V, X¹, Y¹ and Z¹ are the sameor different and each represents hydrogen or a substituent selected fromthe group consisting of hydroxy, halogen, amino, alkoxy, carboxy,aminocarbonylamino, monoalkylaminocarbonylamino,dialkylaminocarbonylamino, carbamato, amido (including formamido,alkylamido and arylamido), aminocarbonylamino,monoalkylaminocarbonylamino, dialkylamino, carboxylamino, carbamato,carboxamido, monoalkylaminosulfinyl, di alkylaminosulfinyl,monoalkylaminosulfonyl, dialkylaminosulfonyl, alkyl sulfonyl amino,hydroxysulfonyloxy, alkoxysulfonyloxy, alkyl sulfonyloxy,hydroxysulfonyl, alkoxysulfonyl, alkyl sulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinyl and, optionallysubstituted, lower alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heterocycle, and heteroaryl; R⁹ is selected from the groups consistingof H, OR¹⁰, and NR¹⁰R¹¹; R¹⁰ and R¹¹ are independently selected from thegroup consisting of H and optionally substituted lower alkyl, alkenyl,aryl, heteroaryl, or heterocycle, or taken together form an optionallysubstituted heterocycle.

In another embodiment, the method of the invention involves theadministration of at least one compound of the formula:

wherein X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, aminocarbonylamino,monoalkylaminocarbonylamino, dialkylaminocarbonylamino, carbamato, amido(including formamido, alkylamido and arylamido), carboxamido,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosulfonyl,dialkylaminosulfonyl, alkyl sulfonyl amino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkylsulfonylalkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl,dialkylaminosulfinyl and, optionally substituted, lower alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heterocycle, and heteroaryl; R¹² is selectedfrom the group consisting of H and optionally substituted lower alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, and heteroaryl;

A is CO and SO₂;

Y is H₂ or O.

In the last-mentioned embodiment, the compound administered may be atleast one of the formula:

wherein X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, amido (including formamido, alkylamidoand arylamido), carboxamido, monoalkylaminosulfinyl,dialkylaminosulfinyl, monoalkylaminosulfonyl, di alkylaminosulfonyl,alkyl sulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy,alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkyl sulfonyl alkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinyl and, optionallysubstituted, lower alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heterocycle, and heteroaryl; R¹² is selected from the group consistingof H and optionally substituted lower alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heterocycle, and heteroaryl; R¹³ is selected from thegroups consisting of H, OR¹⁴, and NR¹⁴R¹⁵; R¹⁴ and R¹⁵ are independentlyselected from the group consisting of H and optionally substituted loweralkyl, alkenyl, aryl, heteroaryl, or heterocycle, or taken together whenattached to a nitrogen atom form an optionally substituted heterocycle;

A is CO and SO₂;

Y is H₂ or O.

In another embodiment of the invention, the compound administered may bea compound of formula VIII:

wherein X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, amido (including formamido, alkylamidoand arylamido), carboxamido, monoalkylaminosulfinyl,dialkylaminosulfinyl, monoalkylaminosulfonyl, di alkylaminosulfonyl,alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy,alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkyl sulfonyl alkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinyl and, optionallysubstituted, lower alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heterocycle, and heteroaryl; R¹⁶ is selected from the group consistingof H and optionally substituted lower alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heterocycle, and heteroaryl; A is CO and SO₂; and Y isH₂ or O. In a preferred embodiment of formula VIII, at least one of X¹,Y¹ and Z¹ is 4-NHCOR¹⁷; wherein R¹⁷ is selected from the groupsconsisting of H, OR¹⁸, and NR¹⁸R¹⁹; R¹⁸ and R¹⁹ are independentlyselected from the group consisting of H and optionally substituted loweralkyl, alkenyl, aryl, heteroaryl, or heterocycle, or taken together whenattached to a nitrogen atom form an optionally substituted heterocycle.

In another aspect, the agents used in the practice of this invention mayhave the general formula:

wherein R²⁰, R²¹ and R²² are independently selected from the groupconsisting of H and optionally substituted lower alkoxy, alkylamino,alkyl, alkenyl, cycloalkyl, aryl, heterocycle, and heteroaryl;

X is O and NR²³; R²³ is selected from the group consisting of H andlower alkyl, or optionally taken together with Q or R¹ and the atoms towhich each is attached forms an optionally substituted 5- or 6-memberedring;

A is CO, CONR²⁴, SO₂, C(═O)—O or a valence bond to R²⁰; R²⁴ is selectedfrom the group consisting of H and lower alkyl;

M is CO and CR²⁵R²⁶; R²⁵ and R²⁶ are independently selected from thegroup consisting of H, lower alkyl, a bond to Q, or optionally takentogether with R²² and the atoms to which each is attached forms anoptionally substituted aryl, heteroaryl, or heterocycle ring;

Q is selected from the group consisting of O, NR²⁷, or a valence bond toR²²; R²⁷ is selected from the group consisting of H, lower alkyl, oroptionally when X is NR²³ and Q is NR²⁷, R²³ and R²⁷ and the nitrogenatom to which each of R²³ and R²⁷ is attached complete an optionallysubstituted 5- or 6-membered heterocycle ring, as represented by—(CR_(a))_(n)—, wherein R_(a) represents H or lower alkyl and n=1 or 2;and the pharmaceutically acceptable salts and all stereoisomers of thecompound.

DESCRIPTION OF DRAWINGS AND TABLES

FIG. 1 sets forth the structures of selected compounds of the invention.Listed compounds show LDLR fold-increase (upregulation) relative tocontrol (no inhibitor) ranging from 1.5 and 30-fold (@25 uM in HEK293cells).

FIG. 2 sets forth an exemplary amino acid sequence for human PCSK9,found as Uniprot Accession Number Q8NBP7 (SEQ ID NO: 1).

FIG. 3 shows the effect of various concentrations of different compoundson PCSK9 processing in HEK293 transfected cells. HEK-293T cells wereseeded into 96 well plates in a DMEM containing 10% Fetal Bovine Serummedia and incubated overnight at 37° C. Cells were transfected withPCSK9 cDNA construct. Compounds (1-50 uM) were added, followed byadditional 43 hours of incubation. Prior to the PCSK9 assay, the cellmedia was replaced with serum free media containing the sameconcentration of compounds or vehicle, and incubated for additional 5hrs. The cell media was analyzed for PCSK9 secretion and cell viabilitydetermined.

FIG. 4 shows the effect of increased degradation of the LDLR by PCSK9.HEK-293T cells were seeded in a DMEM containing 10% Fetal Bovine Serummedia and incubated overnight at 37° C. Cells were transfected with Mock(lanes 1 and 2), PCSK9 (lanes 3 and 4), LDRR & PCSK9 (lanes 5 and 6),LDLR (lanes 7 and 8) cDNA constructs. Cells were incubated foradditional 48 hrs, and cells and media were analyzed as above.

FIG. 5 shows the upregulation of LDLR by PCSK9 antagonists. HEK-293Tcells were seeded in a DMEM containing 10% Fetal Bovine Serum media andincubated overnight at 37° C. Cells were transfected with LDLR & PCSK9cDNA constructs. After 24 hrs, cells were treated with differentconcentration compounds and incubated for additional 48 hrs. Cells werelysed and assayed as described above for LDLR expression.

FIG. 6 shows the effect of specific PCSK9 modulators (400 nMconcentration) on LDLR upregulation in HepG2 cells. PCSK9 transfectedHepG2 cells were seeded into 96 well plates in a MEM containing 10%Fetal Bovine Serum media. Compounds were added, followed by additional43 hours of incubation. The cells were lysed and analyzed for LDLRexpression and cell viability determined as described above.

FIG. 7 shows the increase uptake of Fluorescent Dil-LDL using variousconcentrations of PCSK9 inhibitor in HepG2 cells. The identified SBCcompounds were validated for their ability to increase uptake ofFluorescent Dil-LDL in HepG2 cells. The data show that an increase inthe Fluorescent Dil-LDL uptake using low μM concentrations of SBCcompounds.

FIG. 8 illustrates a general synthesis route for compounds of theFormula III, wherein the diversity elements are introduced in the laststep. The phenyl urea terminus is initially generated by reactingavailable methyl 4-isocyanatobenzoate with ammonia, then aftersaponification the benzoic acid intermediate is converted to an acidchloride then coupled with commercial ethyl mandelate. After a secondsaponification, a library of compounds of the Formula III can begenerated through coupling a set of anilines under standard conditions.

FIG. 9 illustrates a general synthesis route for compounds of theFormulas IV and V, above. The versatile phenyl glycine intermediate(s),3 derived from coupling various anilines to Boc-phenyl glycine, can bebenzoylated with commercial or readily synthesized aryl acid chloridesto provide, after any subsequent elaboration, compounds exemplified bySBC-110,716. Similarly, aryl sulfonylation of intermediate(s), 3provide, after any subsequent elaboration, compounds exemplified bySBC-110,717 and SBC-110,728. Compounds of Formula V can be made usingthe same synthetic route as that for compounds of Formula IV, butreplacing 4-ureido-benzene acid chloride with commercial4-ureido-benzenesulfonyl chloride.

FIG. 10 illustrates a general synthesis route for compounds of theFormula VI (A is CO, SO₂; Y is O). The synthesis begins with commercial3-phenyl-piperazin-2-one. After Boc protection, the first diversityelement, R₁₂ is introduced through an N-arylation cross-couplingreaction with an aryl bromide. After Boc removal, the second diversityelement can be introduced through reaction of available or synthesizedbenzoyl chloride or aryl-sulfonyl chloride derivatives to providecompounds of the Formula VII (e.g., SBC-110,761, FIG. 1).

FIG. 11 illustrates a general synthesis route for compounds of theFormula VII (A is CO, SO₂). The syntheses begin with commercial2-phenyl-piperazine. After selective N-arylation with a substitutedphenyl bromide under cross-coupling conditions to provide intermediate14, reaction with either 4-nitrobenzoyl chloride or 4-nitrobenzenesulfonyl chloride and subsequent nitro reduction provides key anilineintermediates, 15 and 16, respectively. Elaboration of the anilines, 15with sodium cyanate in acetic acid provides mixtures of ureas(SBC-110,733 and SBC-110,734) and acetamides (SBC-110,735, SBC-110,736,and SBC-110,769), while anilines 16 under the same conditions providethe urea (17) and the acetamide (SBC-110,771).

FIG. 12 illustrates a general synthesis route for compounds of theFormula VIII (A is CO) as exemplified by SBC-110,725 and SBC-110,726.The synthesis begins with commercial 2-(piperazin-1-yl)pyrimidine (7).Compounds of the Formula VIII (A is SO₂) can also be prepared using thesame synthesis route, but replacing a benzoic acid analog with abenzenesulfonyl chloride analog in the reaction with 7.

FIG. 13 illustrates a general synthesis route for additional compoundsof the Formula VIII (A is CO). The synthesis begins with commercialBoc-piperazine and its alkylation with a 2-chloromethyl pyridinederivative. After benzoylation with 4-nitrobenzoyl chloride andsubsequent nitro reduction, the aniline can be elaborated into aformamide (SBC-110,729) with sodium cyanate and formic acid, or a urea(12) and an acetamide (SBC-110,730) result when sodium cyanate andacetic acid are used.

FIG. 14 shows the different treatments for each of the five groups ofanimals used to test the efficacy of our compounds.

FIG. 15 shows the effect of compounds on total cholesterol (mg/dl)levels in mice fed high fat diet compared to the control mice.

FIG. 16 shows the percent reduction in total cholesterol levels rangingfrom 18 to 38% when mice fed high fat diet are treated with compoundsand compared to the control mice fed high fat diet.

FIG. 17 shows the effect of combinations of atorvastatin and SBC-110,736on plasma LDL-C in mice fed high fat diet.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to small molecules that down regulate thefunction of extracellular proprotein convertase subtilisin kexin type 9(PCSK9), including its interaction with the low density lipoprotein(LDL) receptor (LDLR), and methods of using these antagonists as amedicament. The small molecule modulators of PCSK9 function can be usedtherapeutically to lower LDL-cholesterol levels in blood, and can beused in the prevention and/or treatment of cholesterol and lipoproteinmetabolism disorders, including familial hypercholesterolemia,atherogenic dyslipidemia, atherosclerosis, and, more generally,cardiovascular disease (CVD).

As used herein, the term “lower alkyl” denotes branched or unbranchedhydrocarbon chains, having 1 to about 8 carbons, such as, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,2-methylpentyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethyl pentyl,octyl, 2,2,4-trimethylpentyl and the like. “Substituted alkyl” includesan alkyl group optionally substituted with one or more functional groupswhich are attached commonly to such chains, such as, hydroxy, halogen,mercapto or thio, cyano, alkylthio, carboxy, carbalkoxy, amino, nitro,alkoxy, or optionally substituted, alkenyl, alkynyl, heterocyclyl, aryl,heteroaryl, and the like to form alkyl groups such as trifluoro methyl,3-hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl, carboxymethyl,cyanobutyl, phenethyl, benzyl and the like.

The term “halogen” or “halo” as used herein alone or as part of anothergroup refers to chlorine, bromine, fluorine, and iodine.

The term “alkoxy” refers to alkyl-O—, in which alkyl is as definedabove.

The term “alkylthio” refers to alkyl-S—, in which alkyl is as definedabove.

The terms “amino”, “monoalkylamino”, “dialkylamino” refers to the moiety—NR′R″, in which R′ and R″, each independently represents H, alkyl oraryl, all as defined herein.

The term “carboxy” refers to the moiety —C(═O)OH.

The term “carbalkoxy” refers to the moiety —C(═O)O-alkyl, in which alkylis as defined above.

The term “carboxamido” refers to the moiety —C(═O)—NR′R″, in which R′and R″, each independently represents H, alkyl or aryl, all as definedherein.

The term “amino (monoalkylamino-, dialkylamino-) carbonylamino” refersto the moiety —NHC(═O)NR′R″, in which R′ and R″ each independentlyrepresents H, alkyl or aryl, all as defined herein.

The term “carbamato” refers to the moiety —NR′C(═O)OR″, in which R′ andR″, each independently represents H, alkyl or aryl, all as definedherein.

The term “amido” refers to the moiety —NRC(═O)—R″, in which R′ and R″,each independently represents H, alkyl or aryl, all as defined herein.

The term “alkylsulfonyl” refers to the moiety —S(═O)₂-alkyl, in whichalkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)₂-alkyl, whereinalkyl is as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfinyl” refers to themoiety —S(═O)NR′R″ in which R′ and R″ each independently represents H,alkyl or aryl, all as defined herein.

The term “amino(monoalkylamino-, dialkylamino-)sulfonyl” refers to themoiety —S(═O)₂NR′R″, in which R′ and R″ each independently represents H,alkyl or aryl, all as defined herein.

The term “alkylsulfonylamino” refers to the moiety —NHS(═O)₂-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyloxy” refers to the moiety —OS(═O)₂OH.

The term “alkoyxsulfonyloxy” refers to the moiety —OS(═O)₂O-alkyl, inwhich alkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)₂-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyl” refers to the moiety —S(═O)₂OH.

The term “alkoxysulfonyl” refers to the moiety —S(═O)₂O-alkyl, whereinalkyl is as previously defined.

The term “alkylsulfonylalkyl” refers to the moiety -alkyl-S(═O)₂-alkyl,wherein alkyl (each instance) is as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfonylalkyl” refers tothe moieties -alkyl-S(═O)₂—NR′R″, wherein alkyl is as previouslydefined, and R′ and R″ each independently represents H, alkyl or aryl,all as defined herein.

The term “amino(monoalkylamino-, dialkylamino-)sulfinylalkyl” refers tothe moieties -alkyl-S(═O)—NR′R″, wherein alkyl is as previously defined,and R′ and R″ each independently represents H, alkyl or aryl, all asdefined herein.

Unless otherwise indicated, the term “cycloalkyl” as employed hereinalone or as part of another group includes saturated or partiallyunsaturated (containing 1 or more double bonds) cyclic hydrocarbongroups containing 1 to 3 rings, including monocyclicalkyl, bicyclicalkyland tricyclicalkyl, containing a total of 3 to 20 carbons forming therings, preferably 3 to 10 carbons, forming the ring and which may befused to 1 or 2 aromatic rings as described for aryl, which includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl cyclododecyl and cyclohexenyl.

“Substituted cycloalkyl” includes a cycloalkyl group optionallysubstituted with 1 or more substituents such as halogen, alkyl,substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl, aryloxy,cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino,amino, nitro, cyano, thiol and/or alkylthio and/or any of thesubstituents included in the definition of “substituted alkyl.”

Unless otherwise indicated, the term “alkenyl” as used herein by itselfor as part of another group refers to straight or branched chain of 2 to20 carbons, preferably 2 to 12 carbons, and more preferably 2 to 8carbons in the normal chain, which include one or more double bonds inthe normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-butenyl,4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl,4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl,4,8,12-tetradecatrienyl, and the like. “Substituted alkenyl” includes analkenyl group optionally substituted with one or more substituents, suchas the substituents included above in the definition of “substitutedalkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “alkynyl” as used herein by itselfor as part of another group refers to straight or branched chain of 2 to20 carbons, preferably 2 to 12 carbons and more preferably 2 to 8carbons in the normal chain, which include one or more triple bonds inthe normal chain, such as 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl,3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl,3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl, 4-dodecynyl and the like.“Substituted alkynyl” includes an alkynyl group optionally substitutedwith one or more substituents, such as the substituents included abovein the definition of “substituted alkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “aryl” or “Ar” as employed hereinalone or as part of another group refers to monocyclic and polycyclicaromatic groups containing 6 to 10 carbons in the ring portion (such asphenyl or naphthyl including 1-naphthyl and 2-naphthyl) and mayoptionally include one to three additional rings fused to a carbocyclicring or a heterocyclic ring, such as aryl, cycloalkyl, heteroaryl orcycloheteroalkyl rings or substituted forms thereof.

“Substituted aryl” includes an aryl group optionally substituted withone or more functional groups, such as halo, alkyl, haloalkyl (e.g.,trifluoromethyl), alkoxy, haloalkoxy (e.g., difluoromethoxy), alkenyl,alkynyl, cycloalkyl-alkyl, cycloheteroalkyl, cycloheteroalkylalkyl,aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy,alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, arylalkenyl,aminocarbonylaryl, arylthio, arylsulfinyl, aryl azo, heteroarylalkyl,heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro,cyano, amino, substituted amino wherein the amino includes 1 or 2substituents (which are optionally substituted alkyl, aryl or any of theother substituents mentioned in the definitions), thiol, alkylthio,arylthio, heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylaminocarbonyl, arylaminocarbonyl, aminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, amido, arylcarbonylamino, arylsulfinyl,arylsulfinylalkyl, arylsulfonylamino or aryl sulfonaminocarbonyl and/orany of the alkyl substituents set out herein.

Unless otherwise indicated, the term “heteroaryl” as used herein aloneor as part of another group refers to a 5- or 7-membered aromatic ringwhich includes 1, 2, 3 or 4 hetero atoms such as nitrogen, oxygen orsulfur and such rings fused to an aryl, cycloalkyl, heteroaryl orheterocycloalkyl ring (e.g. benzothiophenyl, indolyl), and includespossible N-oxides. “Substituted heteroaryl” includes a heteroaryl groupoptionally substituted with 1 to 4 substituents, such as thesubstituents included above in the definition of “substituted alkyl”“substituted cycloalkyl” and “substituted aryl”. Substituted heteroarylalso includes fused heteroaryl groups which include, for example,quinoline, isoquinoline, indole, isoindole, carbazole, acridine,benzimidazole, benzofuran, isobenzofuran, benzothiophene,phenanthroline, purine, and the like.

The term “heterocyclo”, “heterocycle” or “heterocyclic ring,” as usedherein alone or as part of another group, represents an unsubstituted orsubstituted stable 5- to 7-membered monocyclic ring system which may besaturated or partially unsaturated, and which consists of carbon atomsand from one to four heteroatoms selected from N, O or S, and whereinthe nitrogen and sulfur heteroatoms may optionally be oxidized, and thenitrogen heteroatom may optionally be quaternized. The heterocyclic ringmay be attached at any heteroatom or carbon atom which results in thecreation of a stable structure. Examples of such heterocyclic groupsinclude, but are not limited to, piperidinyl, piperazinyl,oxopiperazinyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl,pyrrolyl, pyrrolidinyl, furanyl, thienyl, pyrazolyl, pyrazolidinyl,imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isooxazolyl,isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,thiadiazolyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinyl sulfone, and oxadiazolyl.

The term “optionally substituted” is used herein to signify that achemical moiety referred to, e.g., alkyl, aryl, heteroaryl, may beunsubstituted or substituted with one or more groups including, withoutlimitation, lower alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl, aryl,heterocycle, heteroaryl, hydroxyl, amino, alkoxy, halogen, carboxy,carbalkoxy, carboxamido, amido (including formamido, alkylamido andarylamido), aminocarbonylamino, monoalkylaminocarbonylamino,dialkylaminocarboxylamino, carbamato, monoalkylaminosulfinyl,dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl,alkyl sulfonyl amino, hydroxysulfonyloxy, alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl and the like. Thechemical moieties of the above-described Formulas I-IX which may beoptionally substituted include lower alkyl, alkenyl, alkynyl,cycloalkyl, arylalkyl, aryl, heterocycle, and heteroaryl. For example,optionally substituted alkyl would comprise both propyl and2-chloro-propyl. Additionally, “optionally substituted” is alsoinclusive of embodiments where the named substituent or substituentshave multiple substituents rather than simply a single substituent. Forexample, optionally substituted aryl may comprise both phenyl and3-bromo-4-chloro-6-ethyl-phenyl.

As used herein, the term “subject” includes both humans and animals. Asused herein, the term “PCSK9” refers to any form of the protein PCSK9,including PCSK9 mutants and variants, which retain at least part ofPCSK9 activity or function. Unless otherwise indicated, such as byspecific reference to human PCSK9, PCSK9 refers to all mammalian speciesof native sequence PCSK9, e.g., human, porcine, bovine, equine, canineand feline. One exemplary human PCSK9 sequence is found as UniprotAccession Number Q8NBP7 (SEQ ID NO: 1 (FIG. 2)).

As used herein, a “modulator of PCSK9 function” refers to a smallmolecule that is able to inhibit PCSK9 biological activity or function,and/or downstream pathway(s) mediated by PCSK9 signaling, includingPCSK9-mediated down-regulation of the LDLR, and PCSK9-mediatedinhibition of the decrease in LDL blood clearance. A modulator of PCSK9function encompasses compounds that block, antagonize, suppress orreduce (to any degree including significantly) PCSK9 biologicalactivity, including downstream pathways mediated by PCSK9 signaling,such as LDLR interaction and/or elicitation of a cellular response toPCSK9. For purpose of the present invention, it will be explicitlyunderstood that the term “modulator of PCSK9 function” encompasses allthe previously identified terms, titles, and functional states andcharacteristics whereby the PCSK9 itself, a PCSK9 biological activity(including but not limited to its ability to mediate any aspect ofinteraction with the LDLR, down regulation of LDLR, and inhibit thedecrease in blood LDL clearance), or the consequences of the biologicalactivity, are substantially nullified, decreased, or neutralized in anymeasurable degree. In some embodiments, a modulator of PCSK9 functionbinds PCSK9 and prevents its interaction with the LDLR or its secretion.In other embodiments, a modulator of PCSK9 function binds to the activesite of PCSK9 to stabilize its zymogen and prevent autoprocessing. Infurther embodiments, a modulator of PCSK9 function decreases or blocksPCSK9 mediated down-regulation of the LDLR; inhibits the PCSK9-mediateddecrease in LDL blood clearance; increases LDL clearance in media bycultured hepatocytes; increases blood LDL clearance by the liver invivo; improves patients' sensitivity to other LDL lowering drugs,including statins; is synergistic to other LDL lowering drugs, includingstatins; and blocks PCSK9 interaction with other yet to be identifiedfactors. Examples of modulators of PCSK9 function are provided herein.

The compounds used in the method of the invention can be administered assalts, which are also within the scope of this invention.Pharmaceutically acceptable (i.e., non-toxic, physiologicallycompatible) salts are preferred. If the compounds of the method of thepresent invention have, for example, at least one basic center, they canform acid addition salts. These are formed, for example, with stronginorganic acids, such as mineral acids, for example sulfuric acid,phosphoric acid or a hydrohalic acid, with strong organic carboxylicacids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which areunsubstituted or substituted, for example, by halogen, for exampleacetic acid, such as saturated or unsaturated dicarboxylic acids, forexample oxalic, malonic, succinic, maleic, fumaric, phthalic orterephthalic acid, such as hydroxycarboxylic acids, for exampleascorbic, glycolic, lactic, malic, tartaric or citric acid, such asamino acids, for example aspartic or glutamic acid or lysine orarginine, or benzoic acid, or with organic sulfonic acids, such as(C1-C4) alkyl or arylsulfonic acids which are unsubstituted orsubstituted, for example by halogen, for example methyl- orpara-toluene-sulfonic acid. Corresponding acid addition salts can alsobe formed having plural basic centers, if desired. The compounds used inthe method of the present invention having at least one acid group (forexample COOH) can also form salts with suitable bases. Representativeexamples of such salts include metal salts, such as alkali metal oralkaline earth metal salts, for example sodium, potassium or magnesiumsalts, or salts with ammonia or an organic amine, such as morpholine,thiomorpholine, piperidine, pyrrolidine, a mono, di or tri-loweralkylamine, for example ethyl, tert-butyl, diethyl, diisopropyl,triethyl, tributyl or dimethyl-propylamine, or a mono, di or trihydroxylower alkylamine, for example mono, di or triethanolamine. Correspondinginternal salts may also be formed.

Preferred salts of the compounds described herein which contain a basicgroup include monohydrochloride, hydrogensulfate, methanesulfonate,phosphate or nitrate.

Preferred salts of the compounds described herein which contain an acidgroup include sodium, potassium and magnesium salts and pharmaceuticallyacceptable organic amines.

All stereoisomers of the compounds which may be used in the methodsdescribed herein, either in a mixture or in pure or substantially pureform, are considered to be within the scope of this invention. Thecompounds of the present invention can have asymmetric centers at any ofthe carbon atoms including any one of the R substituents. Consequently,compounds used in the method of the invention can exist in enantiomericor diastereomeric forms or in mixtures thereof. The processes forpreparation of such compounds can utilize racemates, enantiomers ordiastereomers as starting materials. When diastereomeric or enantiomericproducts are prepared, they can be separated by conventional methods forexample, chromatographic, chiral HPLC or fractional crystallization.

As used herein, the term “pharmacophore” refers to the ensemble ofsteric and electronic features that are necessary to ensure the optimalsupramolecular interactions with a specific biological target structureand to trigger, activate, block, inhibit or modulate the biologicaltarget's biological activity, as the case may be. See, IUPAC, Pure andApplied Chemistry (1998) 70: 1129-1143.

As used herein, the term “pharmacophore model” refers to arepresentation of points in a defined coordinate system wherein a pointcorresponds to a position or other characteristic of an atom or chemicalmoiety in a bound conformation of a ligand and/or an interactingpolypeptide, protein, or ordered water molecule. An ordered watermolecule is an observable water in a model derived from structuraldetermination of a polypeptide or protein. A pharmacophore model caninclude, for example, atoms of a bound conformation of a ligand, orportion thereof. A pharmacophore model can include both the boundconformations of a ligand, or portion thereof, and one or more atomsthat interact with the ligand and are from a bound polypeptide orprotein. Thus, in addition to geometric characteristics of a boundconformation of a ligand, a pharmacophore model can indicate othercharacteristics including, for example, charge or hydrophobicity of anatom or chemical moiety. A pharmacophore model can incorporate internalinteractions within the bound conformation of a ligand or interactionsbetween a bound conformation of a ligand and a polypeptide, protein, orother receptor including, for example, van der Waals interactions,hydrogen bonds, ionic bonds, and hydrophobic interactions. Apharmacophore model can be derived from two or more bound conformationsof a ligand.

As used herein, the term “ligand” refers to any compound, composition ormolecule that interacts with the ligand binding domain of a receptor andmodulates its activity. A “ligand” may also include compounds thatmodulate the receptor without binding directly to it.

In carrying out the method of the invention, the above-describedcompounds may be administered as such, or in a form from which theactive agent can be derived, such as a prodrug. A prodrug is aderivative of a compound described herein, the pharmacologic action ofwhich results from the conversion by chemical or metabolic processes invivo to the active compound. The term “prodrug esters” as employedherein includes esters and carbonates formed by reacting one or morehydroxyls of compounds used in the method of the invention with alkyl,alkoxy, or aryl substituted acylating agents employing procedures knownto those skilled in the art to generate acetates, pivalates,methylcarbonates, benzoates and the like. Any compound that can beconverted in vivo to provide the bioactive agent (i.e., a compound offormula I) is a prodrug within the scope and spirit of the invention.Various forms of prodrugs are well known in the art. A comprehensivedescription of prodrugs and prodrug derivatives are described in: (a)The Practice of Medicinal Chemistry, Camille G. Wermuth et al., Ch 31,(Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard,(Elsevier, 1985); (c) A Textbook of Drug Design and Development, P.Krogsgaard-Larson and H. Bundgaard, eds., Ch. 5, pgs, 113-191 (HarwoodAcademic Publishers, 1991).

The therapeutic agent used in practicing the method of the invention isadministered in an amount sufficient to induce the desired therapeuticeffect in the recipient thereof. Thus the term “therapeuticallyeffective amount” as used herein refers to an amount of a therapeuticagent which is sufficient to treat or prevent a condition treatable byadministration of one or more of the compounds of Formulas I-IX, above,or a prodrug thereof. Preferably, the therapeutically effective amountrefers to the amount appropriate to treat a PCSK9-associated condition,i.e. to bring about a detectable therapeutic or preventative orameliorative effect. The effect may include, for example, treatment orprevention of the conditions described herein.

The compound(s) described herein may be administered at a dose in rangefrom about 0.01 mg to about 200 mg/kg of body weight per day. A dose offrom 0.1 to 100, and preferably from 1 to 30 mg/kg per day in one ormore applications per day should be effective to produce the desiredresult. By way of example, a suitable dose for oral administration wouldbe in the range of 1-30 mg/kg of body weight per day, whereas a typicaldose for intravenous administration would be in the range of 1-10 mg/kgof body weight per day. Of course, as those skilled in the art willappreciate, the dosage actually administered will depend upon thecondition being treated, the age, health and weight of the recipient,the type of concurrent treatment, if any, and the frequency oftreatment. Moreover, the effective dosage amount may be determined byone skilled in the art on the basis of routine empirical activitytesting to measure the bioactivity of the compound(s) in a bioassay, andthus establish the appropriate dosage to be administered.

The compounds used in the method of the invention will typically beadministered from 1-4 times a day, so as to deliver the above-mentioneddaily dosage. However, the exact regimen for administration of thecompounds described herein will necessarily be dependent on the needs ofthe individual subject being treated, the type of treatment administeredand the judgment of the attending medical specialist.

In one aspect, the invention provides a method for treating orpreventing hypercholesterolemia, and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease, in anindividual comprising administering to the individual an effectiveamount of a modulator of PCSK9 function that antagonizes circulatingPCSK9.

In a further aspect, the invention provides an effective amount of amodulator of PCSK9 function that antagonizes circulating PCSK9 for usein treating or preventing hypercholesterolemia, and/or at least onesymptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease,in an individual. The invention further provides the use of an effectiveamount of a modulator of PCSK9 function that antagonizes extracellularor circulating PCSK9 in the manufacture of a medicament for treating orpreventing hypercholesterolemia, and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease, in anindividual.

The methods of the invention use a modulator of PCSK9 function, whichrefers to any molecule that blocks, suppresses or reduces (includingsignificantly reduces) PCSK9 biological activity, including downstreampathways mediated by PCSK9 signaling, such as elicitation of a cellularresponse to PCSK9.

A modulator of PCSK9 function should exhibit any one or more of thefollowing characteristics: (a) bind to PCSK9; (b) decrease or blockPCSK9 interaction with the LDLR; (c) decrease or block secretion ofPCSK9; (d) decrease or block PCSK9 mediated down-regulation of the LDLR;(e) inhibit the PCSK9-mediated decrease in LDL blood clearance, (f)increase LDL clearance in media by cultured hepatocytes, (g) increaseblood LDL clearance by the liver in vivo, (h) improve patients'sensitivity to other LDL lowering drugs, including statins, (i) issynergistic to other LDL lowering drugs, including statins; and (j)block PCSK9 interaction with other yet to be identified factors.

In general, the compound(s) used in the method of the invention can beadministered to achieve modulation of PCSK9 function by using anyacceptable route known in the art, either alone or in combination withone or more other therapeutic agents. Thus, the active agent(s) can beadministered orally, buccally, parenterally, such as by intravenous orintra-arterial infusion, intramuscular, intraperitoneal, intrathecal orsubcutaneous injection, by liposome-mediated delivery, rectally,vaginally, by inhalation or insufflation, transdermally or by oticdelivery.

The orally administered dosage unit may be in the form of tablets,caplets, dragees, pills, semisolids, soft or hard gelatin capsules,aqueous or oily solutions, emulsions, suspensions or syrups. Suitabledosage forms for parenteral administration include injectable solutionsor suspensions, suppositories, powder formulations, such asmicrocrystals or aerosol spray. The active agent may also beincorporated into a conventional transdermal delivery system.

As used herein, the expression “physiologically compatible carriermedium” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface agent agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants, fillers and the like as suited for the particular dosageform desired. Remington: The Science and Practice of Pharmacy, 20^(th)edition, (A. R. Genaro et al., Part 5, Pharmaceutical Manufacturing, pp.669-1015 (Lippincott Williams & Wilkins, Baltimore, Md./Philadelphia,Pa.) (2000)) discloses various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Except insofar as any conventional pharmaceutical carriermedium is incompatible with the PCSK9 modulators used in the presentinvention, such as by producing an undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of a formulation comprising such compounds, its use iscontemplated to be within the scope of this invention.

For the production of solid dosage forms, including hard and softcapsules, the therapeutic agent may be mixed with pharmaceuticallyinert, inorganic or organic excipients, such as lactose, sucrose,glucose, gelatine, malt, silica gel, starch or derivatives thereof,talc, stearic acid or its salts, dried skim milk, vegetable, petroleum,animal or synthetic oils, wax, fat, polyols, and the like. For theproduction of liquid solutions, emulsions or suspensions or syrups onemay use excipients such as water, alcohols, aqueous saline, aqueousdextrose, polyols, glycerine, lipids, phospholipids, cyclodextrins,vegetable, petroleum, animal or synthetic oils. For suppositories onemay use excipients, such as vegetable, petroleum, animal or syntheticoils, wax, fat and polyols. For aerosol formulations, one may usecompressed gases suitable for this purpose, such as oxygen, nitrogen andcarbon dioxide. The pharmaceutical composition or formulation may alsocontain one or more additives including, without limitation,preservatives, stabilizers, e.g., UV stabilizers, emulsifiers,sweeteners, salts to adjust the osmotic pressure, buffers, coatingmaterials and antioxidants.

The present invention further includes controlled-release,sustained-release, or extended-release therapeutic dosage forms foradministration of the active agent, which involves incorporation of theactive agent into a suitable delivery system. This dosage form controlsrelease of the active agent(s) in such a manner that an effectiveconcentration of the active agent(s) in the bloodstream may bemaintained over an extended period of time, with the concentration inthe blood remaining relatively constant, to improve therapeutic resultsand/or minimize side effects. Additionally, a controlled-release systemwould provide minimum peak to trough fluctuations in blood plasma levelsof the active agent.

In pharmaceutical compositions used in practicing the method of theinvention, the active agent(s) may be present in an amount of at least0.5 and generally not more than 95% by weight, based on the total weightof the composition, including carrier medium and/or supplemental activeagent(s), if any. Preferably, the proportion of active agent(s) variesbetween 30-90% by weight of the composition.

Preferred compounds for use in practicing this invention include thoseof Formulas II and VI, above. More preferred compounds are those ofFormulas III-V and VII, above. Most preferred are the compounds set outin FIG. 1.

The methods of the present invention will normally include medicalfollow-up to determine the therapeutic or prophylactic effect broughtabout in the subject undergoing treatment with the compound(s) and/orcomposition(s) described herein.

The activities of compounds described herein have been experimentallydemonstrated. The following examples are provided to describe theinvention in further detail. These examples are provided forillustrative purposes only and are not intended to limit the inventionin any way.

Example 1 Test for Secreted PCSK9

HEK-293T cells were seeded into 96-well plates in a DMEM containing 10%Fetal Bovine Serum media and incubated overnight at 37° C. Cells weretransfected with PCSK9 cDNA construct. Compounds at differentconcentrations were added, followed by additional 43 hours ofincubation. Prior to the PCSK9 assay, the cell media was replaced withthe DMEM serum free media containing the same concentration of compoundsor vehicle, and incubated for additional 5 hrs. The cell media wasanalyzed for PCSK9 secretion using western blot analysis, imaged andquantitated using a LAS-4000 (GE). Results from selected compounds areshown in FIG. 3.

Example 2 Test for LDLR Upregulation

A proprietary recombinant assay was used to demonstrate thatco-expression of PCSK9 and LDLR DNA in HEK-293 cells results in adecrease in the expression level of intracellular LDLRs. The presentinventors constructed the expression vector of human LDLR under thecontrol of the cytomegalovirus promoter-enhancer (pCMV-LDLR). Inaddition, a construct containing the PCSK9 (pCMV-PCSK9-FLAG) was made.These constructs were used to transfect mammalian cells and both celllysate and supernatant were subjected to SDS-PAGE and immunoblotanalysis using an anti-PCSK9 or LDLR antibody. The data from the blotshowed that cells that were transfected with only pCMV-PCSK9-FLAGexpressed both the unprocessed (cells) and processed (media) PCSK9 (FIG.4). Cells that were transfected with only pCMV-LDLR showed expression ofthe LDLR in the cells (FIG. 4). However, cells that were transfectedwith both pCMV-PCSK9-FLAG and pCMV-LDLR showed disappearance of theintracellular LDLR band (FIG. 4), which provides further evidence thatthe presence of PCSK9 results in degradation of LDLR or chaperon it tothe degradation pathway. Addition of inhibitors of PCSK9 processing tothe latter cells should result in decreased degradation of the LDLR andthe appearance of the 160K Dalton band on the gel. Using this assay, wetested our compounds for their ability to reduce the degradation of theLDLR. HEK-293 cells were used in this assay. They were grown in 96-wellplates overnight, and transfected with LDLR/PCSK9. Compounds dissolvedin DMSO or vehicle were added to the culture media, and incubated for24-48 hours; cells will then be lysed. Cell lysate were subjected toquantitation using the above immunoassay. Compounds that inhibit thesecretion of the PCSK9 into the media and increase the upregulation ofLDLR were selected (FIG. 5).

Testing confirmed that compounds described herein are capable of upregulating the endogenously expressed LDLR in HepG2 cells. HepG2transfected with PCSK9 cells were cultured in 96-well plates at adensity of 30,000 cells per well. The next day, cells are treated withselected screening compounds or vehicle. Cells were incubated for 48 hrsand then subjected to quantitation using an LDL receptor-polyclonalantibody and analyzed as described above. The data in FIG. 5 show thatthese compounds exhibited an increase in the level of LDLR as comparedto cells treated with same volume of DMSO with several fold upregulationof LDLR at 0.4 uM as compared to control. Thus, the data support ourinitial hypothesis in that an inhibition of the processing and secretionof the PCSK9 results in the upregulation of the LDLR (FIG. 6).

Example 3 Uptake of Dil-LDL in HepG2 Cells In Situ

We also tested the ability of the PCSK9 modulator compounds to enhancethe uptake of Fluorescent DilLDL in HepG2 cells. Briefly, HepG2 cellswere plated and allowed to grow overnight. Compounds were added to thecells followed by the addition of Fluorescent Dil-LDL. Cells were washedextensively, and the Fluorescent Di-LDL taken by the cells were measuredusing the Synergy 2 plate reader (FIG. 7).

Example 4 Test for Cell Viability

All compounds that inhibit PCSK9 secretion will be used to test for insitu cell viability. HEK-293T cells or HepG2 cells were seeded in96-well plates in a cell media containing 10% Fetal Bovine Serum andincubated overnight at 37° C. Compounds at various concentrations willbe added to cells after 24 hours and incubated for an additional 48hours. Cell viability was assayed using Resazurin (Sigma 199303) and aSenergy 2 Multi-label plate reader.

Example 5

General Procedures for Synthesis of Compounds of the Formula III-VIIICompounds of the Formula III, General Procedure for the Preparation ofCompounds of the Formula III (FIG. 8).

Commercial methyl 4-isocyanatobenzoate is reacted with ammonia (slightexcess), then after saponification with LiOH (1.5 equivalents), thecrude acid is directly converted to an acid chloride with oxalylchloride (1.5 equivalents) which is advanced to the next step withoutpurification. The acid chloride is coupled under basic conditions (TEA,2.0 equivalents) with commercial ethyl mandelate (1.5 equivalents) and,after a second saponification with LiOH (3.0 equivalents), the resultingacid is coupled with substituted anilines (2-5 equivalents) utilizingDIPEA and HATU. After aqueous workup, the resulting residue was subjectto flash chromatography using a MeOH gradient (0-10%) in dichloromethaneor reverse phase chromatography (Acetonitrile/water 5-95%) to afford thetarget compounds of the Formula III (e.g., SBC-110,686, FIG. 1).

Compounds of the Formula IV and V.

N-Boc-Phenylglycine (compound 2, FIG. 9) prepared according to theliterature.¹ ¹ Organic Letters, 2004, 6(21), 3675-3678

General procedure for the preparation of compound 3 (FIG. 9):N-Boc-Phenylglycine (cmpd 1; 1 equivalent), the corresponding aniline(1.2 equivalents), N,N-Diisopropylethylamine (5 equivalents) and HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, 1.5 equivalents) were stirred over night at roomtemperature in DMF (dimethylformamide, 1M solution in the starlingmaterial). Following aqueous work up, dichloromethane extraction andevaporation of volatiles, the crude material was flash chromatographedon silica gel columns, using ethyl acetate gradient in hexanes. Theresulting N-Boc-amine was stirred at room temperature in dichloromethane(0.51\4) and excess trifluoroacetic acid (15 equivalents). Afterremoving volatiles the resulting intermediate amine was advanced to thenext step without further purification.

General procedure for the preparation of intermediates 5 and 6 (FIG. 9):Intermediate amine 3 was stirred over night at room temperature indichloromethane with N,N-Diisopropylethylamine (5 equivalents) and4-nitrobenzoyl chloride (1.2 equivalents) or 4-nitrobenzenesulfonylchloride (1.2 equivalents) respectively. Aqueous workup was followed bydichloromethane extraction and removal of volatiles. Without furtherpurification, the crude intermediates were diluted in methanol (0.05M)and aqueous 6N hydrochloric acid solution (10% by volume to themethanol). Hydrogenation was done overnight under hydrogen atmosphereover Pd/C (10%, 0.1 equivalents). The reaction mixture was filtered overCelite® and removal of volatiles afforded crude intermediates 5 and 6which were advanced to the next step without further purification.

Compounds of the Formula VI. General Procedure for the Preparation ofCompounds of the Formula VI (FIG. 10).

Commercial 3-phenyl-piperazin-2-one was protected with Boc₂O (1.5equivalents). After purification, N-arylation was achieved through across-coupling reaction with an aryl bromide (1.2 equivalents), Pd₂dba₃(tris(dibenzylideneacetone)dipalladium(0), 0.2 equivalents), BINAP(2,2′-bis(diphenylphosphino)-1,1′-binaphthyl, 0.2 equivalents), andKOtBu (1.5 equivalents) that were stirred in toluene (0.5M) at 80° C.for 2 hours. After aqueous workup and extraction with dichloromethane,the resulting mixture was flash chromatographed on silica gel using anethyl acetate gradient (0-15%) in hexanes, to afford N-arylpiperazinones.

After Boc removal (TFA, dichloromethane), the amines were advancedwithout further purification. Reaction of the resulting amines withavailable or synthesized benzoyl chlorides or arylsulfonyl chloridederivatives provided, after aqueous workup, a residue that was subjectto flash chromatography using a MeOH gradient (0-5%) in dichloromethaneor reverse phase chromatography (Acetonitrile/water 5-95%) to afford thetarget compounds of the Formula VI.

Compounds of the Formula VII and VIII.

Procedure for the preparation of piperazine 14 (FIG. 11). Commerciallyavailable 2-phenyl-piperazine (1 equivalent), phenyl bromides 13 (1.1equivalents), Pd₂dba₃ tris(dibenzylideneacetone)dipalladium(0), 0.2equivalents), BINAP (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl, 0.2equivalents) and KOtBu (1.5 equivalents) were stirred in toluene (0.5M)at 80° C. for 2 hours. After aqueous workup and extraction withdichloromethane, the resulting mixture was flash chromatographed onsilica gel using an ethyl acetate gradient (0-25%) in hexanes, to affordN-substituted piperazine 14.

General procedure for the preparation of intermediates 8 (FIG. 12), 11(FIG. 13), and 15 and 16 (FIG. 11): Piperazines 7, 10 and 14 werestirred over night at room temperature in dichloromethane withN,N-Diisopropylethylamine (5 equivalents) and 4-nitrobenzoyl chloride(1.2 equivalents) or 4-nitrobenzenesulfonyl chloride (1.2 equivalents),respectively. Aqueous workup was followed by dichloromethane extractionand removal of volatiles. Without further purification, the crudeintermediates were diluted in methanol (0.05M) and aqueous 6Nhydrochloric acid solution (10% by volume to the methanol).Hydrogenation was done overnight under hydrogen atmosphere over Pd/C(10%, 0.1 equivalents). The reaction mixture was filtered over Celite®and removal of volatiles afforded crude intermediates 8, 11, 15 and 16which were advanced to the next step without further purification.

General procedure for the preparation of SBC-110,716, SBC-110,717,SBC-110,728, SBC-110,725, SBC-110,726, SBC-110,729, SBC-110,730,SBC-110,733, SBC-110,734, SBC-110,735, SBC-110,736, SBC-110,769, andSBC-110,771: Intermediates 5, 6, 8, 11, 15 and 16 were stirred overnightwith NaOCN (2 equivalents) in acetic acid and water (10:1, 0.05-0.1M).The reaction mixture was transferred to loose silica gel and volatileswere removed and the residue dissolved in DMSO. Flash chromatographyusing a MeOH gradient (0-10%) in dichloromethane or reverse phasechromatography (Acetonitrile/water 5-95%) afforded the target compounds.

Example 6 Synthesis of SBC-110,716, SBC-110,717 and SBC-110,728

2-Amino-N-(3-chloro-4-methylphenyl)-2-phenylacetamide (compound 3, FIG.9; R=3-Cl-4-Me): N-Boc-Phenylglycine (1.73 g, 6.90 mmol),3-Cl-4-Me-aniline (1.00 mL, 8.28 mmol), diisopropylethylamine (6.00 mL,34.5 mmol) and HATU (3.9 g, 10.35 mmol) were stirred over night in DMF(5 mL) at room temperature. Aqueous work up was followed bydichloromethane extraction and removal of volatiles. Flashchromatography using ethyl acetate gradient (0-35%) in hexanes afforded1.34 g N-Boc protected amine. The compound was stirred overnight withtrifluoroacetic acid (5 mL, excess) in dichloromethane (5 mL). Removalof volatiles afforded 0.99 g of amine 3 (R=3-Cl-4-Me) (97%) which wasadvanced to the next step without further purification. ¹H-NMR (DMSO-d6,400 MHz, δ ppm) 10.77 (s, 1H), 8.77 (s, 1H), 7.75 (d, J=2 Hz, 1H),7.59-7.57 (m, 2H), 7.51-7.45 (m, 3H), 7.36 (dd, J=8.4, 2 Hz, 1H), 7.31(d, J=8.4 Hz, 1H), 5.10 (s, 1H), 2.27 (s, 3H).

4-amino-N-(2-((2-bromo-4-methylphenyl)amino)-2-oxo-1-phenylethyl)benzamide(5): 2-amino-N-(2-bromo-4-methylphenyl)-2-phenylacetamide (0.1 g, 0.31mmol), diisopropylethylamine (0.13 mL, 0.93 mmol) and4-nitro-benzoylchloride (0.07 g, 0.37 mmol) were stirred overnight inDCM (3 mL). Aqueous work up and dichloromethane extraction were followedby flash chromatography using ethyl acetate gradient (0-15%) in hexanes,affording 0.06 g (41%) of the intermediate compound. The intermediateand Pd/C-10% (0.05 g) in methanol (5 mL) and HCl aqueous 6N solution(0.5 mL) were stirred overnight under hydrogen. The reaction mixture wasfiltered over Celite and volatiles were removed. The crude product 0.058g (quantitative yield) was advanced to the next step without furtherpurification. ¹H-NMR (CD₃OD, 400 MHz, δ ppm) 7.91 (d, J=8.8 Hz, 2H),7.49-7.47 (m, 2H), 7.34-7.25 (m, 7H), 7.00 (d, J=8 Hz, 2H), 5.71 (s,1H), 2.18 (s, 3H). See general procedure (Example 5) for sodium cyanatereaction.

SBC-110,716: ¹H-NMR (CD₃OD, 400 MHz, δ ppm) 7.73 (d, J=6.8 Hz, 2H), 7.48(d, J=7.6 Hz, 2H), 7.40 (d, J=6.8 Hz, 2H), 7.35-7.25 (m, 5H), 7.02 (d,J=8.4 Hz, 2H), 5.71 (s, 1H), 2.20 (s, 3H); LC-MS (ESI) (m/z) 481.34.

See general procedure (Example 5) for sulfonylation of 3, followed byhydrogenation and subsequent reaction of intermediate 6 with NaOCN inAcOH.

SBC-110,717: ¹H-NMR (CD₃OD, 400 MHz, δ ppm) 7.60 (d, J=8.8 Hz, 2H),7.35-7.32 (m, 3H), 7.26-7.16 (m, 5H), 7.10-6.94 (m, 3H), 4.90 (s, 1H),2.18 (s, 3H); LC-MS (ESI) (m/z) 472.00.

SBC-110,728: ¹H-NMR (CD₃OD, 400 MHz, δ ppm) 7.70 (d, J=8 Hz, 2H), 7.62(bs, 1H), 7.44 (d, J=8.8 Hz, 2H), 7.36-7.26 (m, 6H), 7.14 (bs, 2H), 5.00(s, 1H), 2.31 (s, 3H); LC-MS (ESI) (m/z) 517.00.

Example 7 Synthesis of SBC-110,725 and SBC-110,726

See general procedure (Example 5) for synthesis of intermediate 8 andsubsequent reaction with NaOCN in AcOH (FIG. 12).

SBC-110,725: ¹H-NMR (DMSO-d6, 400 MHz, δ ppm) 8.76 (s, 1H), 8.39 (d,J=4.8 Hz, 2H), 7.47 (d, J=6.8 Hz, 2H), 7.33 (d, J=6.8 Hz, 2H), 6.67 (t,J=4.8 Hz, 1H), 5.94 (s, 2H), 3.57 (bs, 4H), 3.37 (bs, 4H); LC-MS (ESI)(m/z) 327.10.

SBC-110,726: ¹H-NMR (DMSO-d6, 400 MHz, δ ppm) 10.13 (s, 1H), 8.39 (d,J=4.8 Hz, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.40 (d, J=8.4 Hz, 2H), 6.67 (t,J=4.8 Hz, 1H), 3.78 (bs, 4H), 3.56 (bs, 4H), 2.08 (s, 3H); LC-MS (ESI)(m/z) 326.10.

Example 8 Synthesis of SBC-110,729 and SBC-110,730

Piperazine 10 [1-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)piperazine](FIG. 13): Commercially available N-Boc-piperazine (0.40 g, 2.15 mmol),commercially available 2-chloromethyl-4-methoxy-3,5-dimethylpyridinehydrochloride (0.45 g, 2.03 mmol) and triethylamine (0.85 mmol, 6.10mmol) were stirred overnight at room temperature in dimethylformamide (5mL). After aqueous workup, dichloromethane extraction and removal ofvolatiles, the N-Boc protected intermediate was advanced to the nextstep without further purification. The intermediate was stirred indichloromethane (5 mL) and trifluoroacetic acid (5 mL) overnight at roomtemperature. Aqueous work up was followed by addition of concentratedaqueous NaOH solution (8M) which was added dropwise until the pH wasadjusted to 7. Ethyl acetate extraction and removal of volatilesafforded crude product 10 (0.44 g, 92% over two steps) which wasadvanced to the next step without further purification. ¹H-NMR (CDCl₃,400 MHz, δ ppm) 8.17 (s, 1H), 3.75 (s, 3H), 3.56 (s, 2H), 2.89-2.86 (m,4H), 2.49-2.43 (m, 4H), 2.30 (s, 3H), 2.23 (s, 3H).

SBC-110,729: Intermediate 11 (0.078 g, 0.2 mmol) was stirred overnightwith NaOCN (0.026 g, 0.4 mmol) in formic acid (3 mL). The reactionmixture was transferred to loose silica gel and volatiles were removed.Flash chromatography using a MeOH gradient (0-10%) in dichloromethaneafforded SBC-110,729 0.042 g (55%). NMR spectra shows a 2:1 equilibriumbetween two tautomers: terminal formamide and terminal formimidic acid.¹H-NMR (CD₃OD, 400 MHz, δ ppm) 8.73 (d, J=11.2 Hz, 1H), 8.36 (d, J=1.6Hz, 2H), 8.24 (s, 2H), 8.01 (d, J=10.8 Hz, 1H), 7.90 (s, 2H), 7.55 (d,J=8.8 Hz, 4H), 7.41 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 4H), 7.09 (d,J=8.4 Hz, 2H), 3.80 (s, 12H), 3.68 (s, 8H), 2.60-2.40 (bs, 12H), 2.32(s, 10H), 2.27 (s, 10H); LC-MS (ESI) (m/z) 383.20.

SBC-110,730: See general procedure (Example 5) for reaction ofintermediate 11 and NaOCN in AcOH. ¹H-NMR (CDCl₃, 400 MHz, δ ppm) 8.20(s, 1H), 7.58 (bs, 1H), 7.49 (d, J=8 Hz, 2H), 7.34 (d, J=8.4 Hz, 2H),3.77 (s, 3H), 3.64 (s, 3H), 2.49 (bs, 4H), 2.31 (s, 3H), 2.25 (s, 3H),2.17 (s, 3H), 2.08 (s, 2H); LC-MS (ESI) (m/z) 397.20.

Example 9 Synthesis of SBC-110,733-6, SBC-110,769, and SBC-110,771

1-(3-Chloro-4-methylphenyl)-3-phenylpiperazine (compound of type 14,FIG. 11): 2-phenyl-piperazine (0.13 g, 0.80 mmol), 2-Cl-4-Br-toluene(0.10 mL, 0.76 mmol), Pd₂dba₃ (0.07 g, 0.08 mmol), BINAP (0.048 g, 0.08mmol) and KOtBu (0.13 g, 1.14 mmol) were stirred in toluene (2 mL) at80° C. Aqueous work up, dichloromethane extraction and removal ofvolatiles was followed by flash chromatography separation using ethylacetate gradient (10-60%) in hexanes to afford 0.13 g (56%) of1-(3-Chloro-4-methylphenyl)-3-phenylpiperazine 14. ¹H-NMR (CDCl₃, 400MHz, δ ppm) 7.46-7.44 (m, 2H), 7.39-7.29 (m, 3H), 7.08 (d, J=8.4 Hz,1H), 6.91 (d, J=2.8 Hz, 1H), 6.75 (dd, J=8.8, 2.8 Hz, 1H), 3.96 (dd,J=10.4, 2.8 Hz, 1H), 3.57-3.54 (m, 2H), 3.24 (dt, J=11.6, 3.2 Hz, 1H),2.86 (td, J=11.6, 3.2 Hz, 1H), 2.68 (t, J=11.2 Hz, 1H), 2.27 (s, 3H).

See general procedure (Example 5) for reaction of intermediates 15 and16 with NaOCN in AcOH.

SBC-110,733: ¹H-NMR (CDCl₃, 400 MHz, δ ppm) 7.91 (s, 1H), 7.30 (bs, 2H),7.19 (t, J=7.4 Hz, 2H), 7.13-7-08 (m, 4H), 6.99-6.94 (m, 3H), 6.75 (d,J=2.4 Hz, 1H), 6.58 (dd, J=8.4, 2.4 Hz, 1H), 4.95 (bs, 2H), 3.92 (d,J=12.4 Hz, 1H), 3.32-3.21 (m, 2H), 3.06 (dd, J=12.8, 3.6 Hz, 1H), 2.72(t, J=10.4 Hz, 1H), 2.12 (s, 3H); LC-MS (ESI) (m/z) 449.10.

SBC-110,734: ¹H-NMR (CD₃OD, 400 MHz, δ ppm) 8.48 (s, 1H), 7.45 (bs, 2H),7.42 (d, J=8.4 Hz, 2H), 7.35-7.29 (m, 4H), 7.23-7-19 (m, 1H), 7.00 (d,J=8.4 Hz, 2H), 6.83 (d, J=8.4 Hz, 2H), 4.51 (s, 1H), 4.14 (d, J=12.4 Hz,1H), 3.87-3.84 (m, 1H), 3.45-3.41 (m, 1H), 3.10 (dd, J=12.8, 4 Hz, 1H),2.78 (td, J=11.8, 3.2 Hz, 1H), 2.18 (s, 3H); LC-MS (ESI) (m/z) 415.20.

SBC-110,735: ¹H-NMR (CDCl₃, 400 MHz, δ ppm) 8.00 (bs, 1H), 7.45-7.35 (m,4H), 7.30-7.20 (m, 5H), 7.04 (d, J=8.4 Hz, 1H), 6.85 (d, J=2.4 Hz, 1H),6.68 (dd, J=8.4, 2.4 Hz, 1H), 4.03 (d, J=12.4 Hz, 1H), 3.37 (d, J=10.8Hz, 1H), 3.27 (t, =11.8 Hz, 1H), 3.13 (dd 12.4 Hz, 1H), 2.79 (t, J=10.2Hz, 1H), 2.21 (s, 3H), 2.07 (s, 3H); LC-MS (ESI) (m/z) 448.15.

SBC-110,736: ¹H-NMR (CDCl₃, 400 MHz, δ ppm) 7-56-7.50 (m, 4H), 7.41-7.28(m, 5H), 7.11 (d, J=8 Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 4.14 (d, J=12.4Hz, 1H), 3.45 (d, J=11.2 Hz, 1H), 3.35 (t, J=12.4 Hz, 1H), 3.19 (dd,J=12.8, 4 Hz, 1H), 2.86 (t, J=11 Hz, 1H), 2.29 (s, 3H), 2.18 (s, 3H);LC-MS (ESI) (m/z) 414.20.

SBC-110,769: ¹H-NMR (DMSO-d6, 400 MHz, δ ppm) 10.12 (s, 1H), 7.95 (d,J=8.4 Hz, 1H), 7.80-7.60 (m, 3H), 7.55-7.25 (m, 11H), 7.15 (d, J=7.6 Hz,1H), 3.81-3.79 (m, 2H), 2.87-2.84 (m, 1H), 2.58 (s, 3H), 2.06 (s, 3H);LC-MS (ESI) (m/z) 464.20.

SBC-110,771: ¹H-NMR (DMSO-d6, 400 MHz, δ ppm) 9.06 (s, 1H), 7.92 (d, J=8Hz, 1H), 7.70-7.66 (m, 3H), 7.59 (d, J=8.8 Hz, 2H), 7.53-7.46 (m, 3H),7.37 (t, J=7.4 Hz, 2H), 7.30 (t, J=7.6 Hz, 2H), 7.23 (d, J=7.6 Hz, 1H),6.90 (d, J=7.6 Hz, 1H), 6.10 (s, 2H), 3.82 (d, J=13.6 Hz, 1H), 3.61 (t,J=11.4 Hz, 1H), 3.52 (d, J=12 Hz, 1H), 3.09 (d, J=12.4 Hz, 1H), 2.98(dd, J=12, 4 Hz, 1H), 2.54 (s, 3H), 2.45 (t, J=1.8 Hz, 1H); LC-MS (ESI)(m/z) 500.10.

Example 10 Test for Efficacy in Animal Model

SBC-110,686, SBC-110,733 and SBC-110,736 were tested for their efficacyin male mice (C57BL/6 mice). Mice were housed as four animals per cageunder climate-controlled conditions of temperature (20-24° C.), humidity(60-70%), and alternating 12 h light/dark cycles. The mice were dividedinto five groups as shown in FIG. 14. One group was fed commercial chowdiet (Prolab RMH 3000, PMI feeds, St. Louis, Mo.) to serve as a negativecontrol, while the other four groups were fed high fat diet (TD.06414),which provides 60% of calories from fat. Water was provided ad libitum.Plasma was collected once weekly to monitor the level of LDL. After 4weeks of feeding a high fat diet, mice were randomly assigned to one ofseveral groups such that the average LDL levels were equal amongdifferent groups. One of the four groups of mice fed high fat diet wastreated with vehicle and served as a positive control, whereas each ofthe other three groups was treated daily with 8 mg/kg of one of thecompounds subcutaneously for two weeks. Blood samples (75 μl) werecollected twice weekly after drug administration from the retro-orbitalvenous plexus via heparinized capillary tubes containing 2 USP units ofammonium heparin per tube (Carolina, Burlington, N.C.). Plasma wereseparated immediately by centrifugation (5,000×g) for 5 min at roomtemperature and then kept at −80° C. until assayed for lipid profile.Plasma cholesterol, LDL-C, HDL-C, and triglyceride levels were measuredenzymatically. Additionally, the plasma levels of PCSK9 andchemokines/cytokines were measured for potential pleiotropic effects ofPCSK9 inhibitors using ELISA and multiplex assays.

Our data demonstrated that SBC-110,686, SBC-110,733 and SBC-110,736lowered cholesterol levels in mice that were fed high fat diet (FIGS. 15and 16), with SBC-110,736 showing a mean of 38% reduction (P<0.01) intotal cholesterol levels after two weeks relative to high fat dietanimal levels and a mean 50% reduction (P<0.01) toward return to regulardiet cholesterol levels.

A second study was conducted with atorvastatin. Data demonstrated thatthe compounds in combination with atorvastatin resulted in an additiveeffect on lowering LDL-C levels in mice fed high fat diet. FIG. 17 showsdata obtained from SBC-110,736 in combination with atorvastatin in mice,indicating an additive effect of reduction in LDL-C level after twoweeks of treatments.

The foregoing specification includes citations to certain publications,which are provided to indicate the state of the art to which thisinvention pertains. The entire disclosure of each of the citedpublications is incorporated by reference herein.

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing disclosure. The presentinvention is, therefore, not limited to the particular embodimentsdescribed and/or exemplified, but is capable of considerable variationand modification without departure from the scope of the appendedclaims. Furthermore, the transitional terms “comprising”, “consistingessentially of” and “consisting of”, when used in the appended claims,in original and amended form, define the claim scope with respect towhat unrecited additional claim elements or steps, if any, are excludedfrom the scope of the claim(s). The term “comprising” is intended to beinclusive or open-ended and does not exclude any additional, unrecitedelement, method, step or material. The term “consisting of” excludes anyelement, step or material other than those specified in the claim and,in the latter instance, impurities ordinarily associated with thespecified material(s). The term “consisting essentially of” limits thescope of a claim to the specified elements, steps or material(s) andthose that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. All compositions and methodsof use thereof that embody the present invention can, in alternateembodiments, be more specifically defined by any of the transitionalterms “comprising”, “consisting essentially of” and “consisting of”.

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What is claimed is:
 1. A method for treating or preventinghypercholesterolemia, and/or at least one symptom of dyslipidemia,atherosclerosis, CVD or coronary heart disease in a patient in need ofsaid treatment, the method comprising administering to said patient atherapeutically effective amount of at least one compound of formula(I):

wherein: R¹, R² and R³ are independently selected from the groupconsisting of H and optionally substituted lower alkyl, alkenyl,cycloalkyl, aryl, heterocycle, and heteroaryl; X is O or NR⁴; wherein R⁴is H, lower alkyl, or optionally taken together with Q or R¹ and theatoms to which each is attached forms an optionally substituted 5- or6-membered heterocycle ring; A is CO, CONR⁵, SO₂, C(═O)—O or a valencebond to R¹; wherein R⁵ is H or lower alkyl; M is CO or CR⁶R⁷; wherein R⁶and R⁷ are independently H, lower alkyl, a bond to Q, or optionallytaken together with R³ and the atoms to which each is attached forms anoptionally substituted aryl, heteroaryl, or heterocycle ring; and Q is Oor NR⁸; wherein R⁸ is H, lower alkyl, or optionally when X is NR⁴ and Qis NR⁸, R⁴ and R⁸ and the nitrogen atom to which each of R⁴ and R⁸ isattached complete an optionally substituted 5- or 6-membered heterocyclering, as represented by —(CR_(a))_(n)—, wherein R_(a) represents H orlower alkyl and n=1 or 2; and the pharmaceutically acceptable salts andall stereoisomers of the compound.
 2. The method of claim 1, whereinsaid patient is administered at least one compound of formula (II):

wherein: X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting ofhydroxyl, halogen, amino, alkoxy, carboxy, amido, formamido, alkylamido,arylamido, aminocarbonyl amino, monoalkylaminocarbonylamino,dialkylaminocarbonylamino, carbamato, carboxamido,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkyl sulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkyl sulfonyl alkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl,dialkylaminosulfinyl and, optionally substituted, lower alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heterocycle, and heteroaryl; X is O or NR⁴;wherein R⁴ is selected from the group consisting of H and lower alkyl;and A is CO, CONR⁵, SO₂, C(═O)—O or a valence bond to R¹; wherein R⁵ isH or lower alkyl.
 3. The method of claim 2, wherein said patient isadministered at least one compound of formula (III):

wherein: X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, amido, formamido, alkylamido,arylamido, aminocarbonyl amino, monoalkylaminocarbonylamino,dialkylaminocarbonylamino, carbamato, carboxamido,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkyl sulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkyl sulfonyl alkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl,dialkylaminosulfinyl and, optionally substituted, lower alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heterocycle, and heteroaryl; and R⁹ isselected from the group consisting of H, OR¹⁰, and NR¹⁰R¹¹; wherein R¹⁰and R¹¹ are independently selected from the group consisting of H andoptionally substituted lower alkyl, alkenyl, aryl, heteroaryl, orheterocycle, or taken together form an optionally substitutedheterocycle.
 4. The method of claim 2, wherein said patient isadministered at least one compound of formula (IV):

wherein: X¹, Y¹ and Z¹ are the same or different and each representhydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, amido, formamido, alkylamido,arylamido, aminocarbonyl amino, monoalkylaminocarbonylamino,dialkylaminocarbonylamino, carbamato, carboxamido,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkyl sulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkyl sulfonyl alkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl,dialkylaminosulfinyl and, optionally substituted, lower alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heterocycle, and heteroaryl; and R⁹ isselected from the group consisting of H, OR¹⁰, and NR¹⁰R¹¹; wherein R¹⁰and R¹¹ are independently selected from the group consisting of H andoptionally substituted lower alkyl, alkenyl, aryl, heteroaryl, orheterocycle, or taken together form an optionally substitutedheterocycle.
 5. The method of claim 2, wherein said patient isadministered at least one compound of formula (V):

wherein: X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, carboxamido, monoalkylaminosulfinyl,dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl,alkyl sulfonyl amino, hydroxysulfonyloxy, alkoxysulfonyloxy,alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinyl and, optionallysubstituted, lower alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heterocycle, and heteroaryl; and R⁹ is selected from the groupconsisting of H, OR¹⁰, and NR¹⁰R¹¹; wherein R¹⁰ and R¹¹ areindependently selected from the group consisting of H and optionallysubstituted lower alkyl, alkenyl, aryl, heteroaryl, or heterocycle, ortaken together form an optionally substituted heterocycle.
 6. The methodof claim 1, wherein said patient is administered at least one compoundof formula (VI):

wherein: X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, carboxamido, monoalkylaminosulfinyl,dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl,alkyl sulfonyl amino, hydroxysulfonyloxy, alkoxysulfonyloxy,alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinyl and, optionallysubstituted, lower alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heterocycle, and heteroaryl; R¹² is selected from the group consistingof H and optionally substituted lower alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heterocycle, and heteroaryl; A is CO or SO₂; and Y isH₂ or O.
 7. The method of claim 6, wherein said patient is administeredat least one compound of e formula (VII):

wherein: X¹, Y¹ and Z¹ are the same or different and each representshydrogen or a substituent selected from the group consisting of hydroxy,halogen, amino, alkoxy, carboxy, carboxamido, monoalkylaminosulfinyl,dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl,alkyl sulfonyl amino, hydroxysulfonyloxy, alkoxysulfonyloxy,alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinyl and, optionallysubstituted, lower alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heterocycle, and heteroaryl; R¹² is selected from the group consistingof H and optionally substituted lower alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heterocycle, and heteroaryl; R¹³ is selected from thegroups consisting of H, OR¹⁴, and NR¹⁴R¹⁵; wherein R¹⁴ and R¹⁵ areindependently selected from the group consisting of H and optionallysubstituted lower alkyl, alkenyl, aryl, heteroaryl, or heterocycle, ortaken together when attached to a nitrogen atom form an optionallysubstituted heterocycle; A is CO or SO₂; and Y is H₂ or O.
 8. The methodof claim 2, wherein the compound is selected from the group consistingof [(3-chloro-4-methylphenyl)carbamoyl](phenyl)methyl4-(carbamoylamino)benzoate (SBC-110,686);N-(3-chloro-4-methylphenyl)-2-[(4-acetamidophenyl)formamido]-2-phenylacetamide(SBC-110,720); and [(3-chloro-4-methylphenyl)carbamoyl](phenyl)methyl4-acetamidobenzoate (SBC-110,721).
 9. The method of claim 2, wherein thecompound isN-{4-[4-(4-methylphenyl)-2-phenylpiperazine-1-carbonyl]phenyl}acetamide(SBC-110,736) or{4-[4-(3-chloro-4-methylphenyl)-2-phenylpiperazine-1-carbonyl]phenyl}urea(SBC-110,733).
 10. The method of claim 2, further comprisingadministering to said patient a therapeutically effective amount of alow density lipoprotein (LDL) lowering drug.
 11. The method of claim 10,wherein said LDL lowering drug is a statin.
 12. The method of claim 6,further comprising administering to said patient a therapeuticallyeffective amount of a low density lipoprotein (LDL) lowering drug. 13.The method of claim 12, wherein said LDL lowering drug is a statin.